Spider Silk Protein Production using DNA Recombination
Overview Spider silk is a highly coveted and useful material that can be used for a variety of purposes. It is tougher than kevlar and steel on an equal weight basis, as well as being biocompatible and biodegradable. This leads to spider silk proteins being excellent candidates for tissue engineering, guided tissue repair, and drug delivery. Spider silk can also be used for cosmetic, and industrial purposes. Spider silk production on a large scale has proven difficult due to orb-weaver spiders territorial and cannibalistic nature. Recently, recombinant gene technology has made it possible to use docile transgenic hosts, such as goats, to synthesize spider silk proteins. However, large scale spider silk production has still proven to be challenging. There are at least seven known types of spider silk proteins for one species of orb-weaver spider. The proteins differ in primary structure, physical properties, and function. However, all spider silk proteins consist of three domains: a repetitive middle core that makes up most of the protein chain, and non-repetitive N-terminal and C-terminal ends. The Structure of the Proteins Although one spider can produce up to seven different silk types the basic structure is a repetitive middle core, and non-repetitive N, and C-terminals. The most studied silk type is dragline silk, which is used to build frames, lifelines, and radii for webs. The golden orb-weaver spider produces dragline silk in the major ampullate gland. Dragline silk is a protein complex composed of Major ampullate draglinee silk protein 1 (MaSp 1), and major ampullate draglinee silk protein 2 (MaSp 2). Both proteins are made up of about 3500 amino acids. MaSp 1 can be found in the protein core and on the sides, MaSp2 can only be found at certain areas on the core of the protein. Cloning There are many ways in which to clone the spider silk protein. In the figure to the right a cloning vector, such as a BAC is used, and the gene for the spider silk monomer is cloned and spliced in. The first stem in the process is digestion in which the cloning vector and the gene are digested with restriction enzymes. The next step few steps are all ligations. First, the ligation of the linker segment occurs, then ligation of the silk monomer gene segment occurs, the last ligation step is ligating the silk multimer gene into the cloning vector. Now transformation into a bacteria such as E. coli can occur. Once transformation occurs the next step is purification of the final product. Purification There are many diverse ways to purify spider silk proteins from different organisms, but a widely used technique is IMAC. IMAC is an acronym for Immobilized Metal Affinity Chromatography. In this technique target proteins are tagged with histidine. The tendancy for the histidine in the protein to form a complex with a divalent metal in a neutral pH is used to purify the protein. The metal then sticks to a chromatographic resin separating tagged proteins from untagged proteins. References Tokareva, Olena, Valquíria A. Michalczechen-Lacerda, Elíbio L. Rech, and David L. Kaplan. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 11 Oct. 2013. Web. 21 Sept. 2014. . "Spider Silk." Wikipedia. Wikimedia Foundation, 12 July 2014. Web. 08 Dec. 2014. .